title: Sec-mediated secretion by Coxiella burnetii.
abstract: BACKGROUND
  Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication.
  
  
  RESULTS
  To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase.
  
  
  CONCLUSIONS
  C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA. rheinzen@niaid.nih.gov.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Stead
authors.forename: Anders
authors.initials: A
authors.lastname: Omsland
authors.forename: Paul A
authors.initials: PA
authors.lastname: Beare
authors.forename: Kelsi M
authors.initials: KM
authors.lastname: Sandoz
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019295:Computational Biology; D016997:Coxiella burnetii / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D017384:Sequence Deletion; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D050505:Mutant Proteins; D021382:Protein Sorting Signals